1. Field of the Invention
The present invention relates to selective expression of genes useful in gene therapy protocols via photoactivation. In particular, the present invention provides a means of selectively expressing genes in specific cells, comprising delivering xe2x80x9ccagedxe2x80x9d (inactivated) nucleic acid to cells and xe2x80x9cuncagingxe2x80x9d (activating) the nucleic acid by exposure of the targeted cells to light, thereby allowing temporally controlled expression of exogenous nucleic acid only in targeted cells or selectively regulating endogenous gene expression.
2. Background Art
The as yet unrealized goal of in vivo gene therapy is the expression of exogenous genetic material within only a target cell population. Successful in vivo gene therapy must overcome two challenges: 1) delivery of genes to the specific target cell population and 2) subsequent expression only within these cells. Viral and non-viral technologies for targeted delivery of genes have been evaluated. These include localized injection in skeletal muscle (Manthrope et al., 1993; reviewed in Brown et al., 1996), targeting of liposomes by incorporating antibodies to unique cell surface markers in the liposome outer surface (reviewed in Torchilin, 1996) and use of viruses with naturally selected sub-population targets, such as adenovirus for the bronchial epithelium (Rosenfeld et al., 1993). Although all of these strategies require that each target cell population be uniquely defined, the potential utility has kept interest high. Because of immune responses with adenovirus, safety issues with retroviruses and the poor targeting ability of liposomes, none of these strategies has proven suitable in its current form for targeted delivery and expression of genes.
In addition, targeted post-delivery expression strategies have been attempted (reviewed by Yarranton, 1992). These strategies involve delivery of nucleic acids comprising elements which can be broadly classified into 1) inducers triggered by changes in the cellular environment (cell milieu inducers) and 2) promoters which induce expression only within specific tissues. Cell milieu inducers can include promoters sensitive to metal concentration (Searle et al., 1985; Mayo et al., 1982), tetracycline (Furth et al., 1994; Gossen et al., 1995), hormones (Hynes et al., 1981; Andres et al., 1987) and the insect molting hormone ecdysone (No et al., 1996). However, the cell milieu inducers cannot be used to target sub-populations of cells, since all transfected cells respond to such changes in the cellular environment. Furthermore, unique tissue-specific promoters (reviewed by Hart, 1996; Stein et al., 1996) must be developed for each individual target cell population.
Photosensitive precursors or xe2x80x9ccagingxe2x80x9d groups are molecules which bind an xe2x80x9ceffectorxe2x80x9d molecule through a covalent bond to the photosensitive precursor group, thereby reversibly rendering the effector molecule inert (McCray et al., 1989). The term xe2x80x9ccagedxe2x80x9d is merely descriptive of the photo release property of these groups and does not refer to physical trapping of the inactivated substance within a crystal lattice. Caging groups have been used in a number of biological studies to study cell motility, muscle fibers, active transport proteins, biological membranes and other intracellular responses (e.g. Ishihara et al., 1997; Lee et al., 1997; Patton et al., 1991; see review by McCray et al., 1989). Caging groups have also been used in the caging of nucleotide analogues (Walker et al., 1988) and the synthesis of bio-chip arrays (McGall et al., 1996). Classically, caging groups have been used to study the time course of cellular responses induced by a step change in a local concentration of caged and subsequently, inactivated bio-chemical species, e.g. caged ATP. A rapid localized increase in concentration or activity of the caged substance is achieved by application of a directed pulse of light, which releases the bio-chemical inactivating group and returns the caged species to its biologically active state. In the case of caged ATP, this results in a localized high concentration of ATP. However, neither the caging of nucleic acids for selective regulation of gene expression nor the use of caged nucleic acids in therapeutic applications such as gene therapy have been described.
The present invention overcomes previous shortcomings in gene therapy technology by providing methods whereby caged genes or caged proteins can be delivered nonspecifically to cells and the genes or proteins in selected cells can be activated by exposure to light, thereby limiting the expression of genes or activity of exogenous proteins to selected cells. The methods of this invention can be employed to treat a variety of disease states and genetic disorders.
The present invention provides an isolated nucleic acid covalently linked to a photolabile caging group which reversibly prevents expression of the nucleic acid.
The present invention further provides a method of selectively expressing a nucleic acid in a cell, comprising: a) covalently linking the nucleic acid to a photolabile caging group which reversibly prevents expression of the nucleic acid; b) introducing the nucleic acid of step (a) into the cell; and c) exposing the cell of step (b) to light, whereby exposure to the light unlinks the nucleic acid and the caging group and the nucleic acid is selectively expressed in the cell.
The present invention additionally provides a method of selectively regulating the expression of an endogenous nucleic acid comprising: a) covalently linking a nucleic acid encoding an antisense nucleic acid to a photolabile caging group which reversibly prevents expression of the nucleic acid; b) introducing the nucleic acid of step (a) into the cell; and c) exposing the cell of step (b) to light, whereby exposure to the light unlinks the nucleic acid and the caging group and the nucleic acid is selectively expressed in the cell as an antisense nucleic acid which can bind to and inactivate a complementary nucleic acid within the cell.
Various other objectives and advantages of the present invention will become apparent from the following detailed description.
As used herein, xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d can mean multiples.
The present invention provides a novel strategy for localized targeting of gene expression based on delivering reversibly inactivated genes to cells which can be selectively expressed in targeted cells upon activation of the genes by exposure to light. Transcription of the genes in cells is blocked by biochemical modification of the plasmid nucleic acid with a xe2x80x9ccagingxe2x80x9d group. Activation of transcription is achieved by xe2x80x9cuncagingxe2x80x9d the plasmid by exposure to light.
Thus, the present invention provides an isolated nucleic acid covalently linked to a photolabile caging group which reversibly prevents expression of the nucleic acid. As used herein, xe2x80x9cnucleic acidxe2x80x9d refers to single- or double-stranded molecules which may be DNA, comprising two or more nucleotides comprised of the nucleotide bases A, T, C and G, or RNA, comprised of the bases A, U (substitute for T), C and G. The nucleic acid may represent a coding strand or its complement. Nucleic acids may be identical in sequence to a sequence which is naturally occurring or may include alternative codons which encode the same amino acid as that which is found in a naturally occurring sequence (Lewin, 1994). Furthermore, nucleic acids may include codons which represent conservative substitutions of amino acids as are well known in the art. With regard to gene therapy applications, the nucleic acid can comprise a nucleotide sequence which encodes a gene product which is meant to function in the place of a defective gene product and restore normal function to a cell which functioned abnormally due to the defective gene product. Alternatively, the nucleic acid may encode a gene product which was not previously present in a cell or was not previously present in the cell at a therapeutic concentration, whereby the presence of the exogenous gene product or increased concentration of the exogenous gene product imparts a therapeutic benefit to the cell and/or to a subject. For example, the nucleic acid of this invention can include but is not limited to, a gene encoding a gene product that promotes cell killing, a gene encoding a gene product involved in inherited disorders, a gene encoding a gene product that promotes wound repair, a gene encoding a gene product which promotes cell-cell adhesion and a gene encoding a gene product which modulates cellular signals.
As used herein, the term xe2x80x9cisolatedxe2x80x9d means a nucleic acid separated or substantially free from at least some of the other components of the naturally occurring organism, for example, the cell structural components commonly found associated with nucleic acids in a cellular environment and/or other nucleic acids. The isolation of nucleic acids can therefore be accomplished by techniques such as cell lysis followed by phenol plus chloroform extraction, followed by ethanol precipitation of the nucleic acids (Michieli et al., 1996). The nucleic acids of this invention can be isolated from cells according to methods well known in the art. Alternatively, the nucleic acids of the present invention can be synthesized according to standard protocols well described in the literature.
Also as used herein, xe2x80x9cexpression of the nucleic acidxe2x80x9d means transcription of DNA into RNA, translation of RNA into an amino acid sequence with subsequent modifications to produce a functional polypeptide or both transcription and translation.
The nucleic acid of this invention can be part of a recombinant nucleic acid comprising any combination of restriction sites and/or functional elements as are well known in the art which facilitate molecular cloning, expression and other recombinant DNA manipulations. Thus, the present invention further provides a recombinant nucleic acid comprising the nucleic acid of the present invention. In particular, the nucleic acid can be present in a vector and the vector can be present in a cell, which can be a cell cultured in vitro or a cell in an animal.
Thus, the present invention further provides a vector comprising a nucleic acid of this invention. The vector can also include other amino acid-encoding nucleotide sequences. The vector can be an expression vector which contains all of the genetic components required for expression of the nucleic acid in cells into which the vector has been introduced, as are well known in the art. The expression vector can be a commercial expression vector or it can be constructed in the laboratory according to standard molecular biology protocols. The expression vector can comprise viral nucleic acid including, but not limited to, adenovirus, retrovirus and or adeno-associated virus nucleic acid. The nucleic acid or vector of this invention can also be in a liposome or a delivery vehicle which can be taken up by a cell via receptor-mediated or other type of endocytosis. The nucleic acid or vector of this invention can also be in a pharmaceutically acceptable carrier, as described below.
The caging group of the present invention can be any caging group which is photolabile, i.e., which undergoes a chemical reaction with a target molecule whereby the caging group covalently attaches to the target molecule (Walker et al, 1988), thereby inhibiting the biological activity of the target molecule, and which upon subsequent exposure to a radiation source (e.g., UV wavelength), undergoes a conformational change that breaks the covalent bond to the target molecule and restores the biological activity of the target molecule (e.g., nucleic acid, amino acid sequence).
The caging group of the present invention can but is not limited to, DMNPE 1-(4,5-dimethyoxy-2-nitrophenyl) ethyl, (2- nitrophenyl) ethyl, 5-carboxymethoxy-2-nitrobenzyl, ((5-carboxymethoxy-2-nitrobenzyl)oxy) carbonyl, 4,5-dimethyoxy-2-nitrobenzyl, ((4,5-dimethoxy-2nitrobenzyl)oxy) carbonyl, alpha-carboxy-2-nitrobenzyl, 1-(2-nitrophenyl) ethyl, 2nitrobenzyl and Desoxybenzoinyl, as well as any other photolabile caging group now known or later developed which can covalently attach to a sequence of two or more nucleotides or to an amino acid sequence and reversibly inhibit the biological activity of the molecule or substance to which the caging group is attached. By xe2x80x9creversiblyxe2x80x9d is meant that the inhibitory effect of the caging group can be initiated by binding the caging group to a target molecule or substance and removed by exposing the caged molecule or substance to light, thereby altering the conformation of the caging group and restoring biological activity to the target molecue or substance. The caging groups listed herein are known in the art and can be routinely synthesized or purchased. Additional caging groups having the properties of the caging groups of this invention can be identified according to the protocols described herein and as described in the literature.
The caging group can be covalently bound to a target molecule by combining the caging group with a target molecule after activation of the caging group with an oxidant such as manganese oxide. As a result of the covalent bonding to a caging group, the physical structure of the target molecule is reversibly modified (e.g., the tertiary structure is altered in various ways, which can include coiling, looping and twisting), the bioactivity of the target molecule is reversibly inhibited (e.g., the stability of the target molecule in vivo is increased due to protection against non-specific DNase or RNase enzymatic cleavage as well as protection against site-specific enzymes; and other biological processing molecules which act via recognition of DNA or RNA are blocked) and delivery of the caged molecule to cells is enhanced by greater affinity of binding with cell membranes or by enhanced binding to a delivery vehicle (e.g., liposome).
The type of irradiation for altering the conformation of the caging group on the target molecule and restoring biological activity to the target molecule can be, but is not limited to, light at a wavelength in a range from 300 to 700 nm and more preferably at a wavelength in a range from 300 to 400 mn and most preferably at a wavelength of 365 nm, as well as irradiation at other wavelengths, such as X-rays, magnetic resonance and thermal energy. The source of the irradiation can be any source by which a substance can be exposed to the radiation, including but not limited to, a lamp, a fiber optic device, a laser, an X-ray machine, a light emitting compound which can be ingested and a lithotripsy device which produces light by bubble collapse.
The caged nucleic acid of this invention can be used to target localized gene expression to a specific cell population after non-localized gene delivery to cells as well as to target localized blockade of normal expression pathways by caged anti-sense nucleic acids. In addition, the caged nucleic acid can be used in vitro as a light sensitive trigger for activation of the polymerase chain reaction (PCR) as an alternative to xe2x80x9chot startxe2x80x9d PCR, for control of primer concentrations in PCR (e.g., light-activated nested PCR), for sequencing of DNA or RNA by differential bio-activation of 5xe2x80x2 or 3xe2x80x2 ends to allow selective cleavage of end nucleotides and for sequential activation using two or more different caging groups which are triggered to xe2x80x9cuncagexe2x80x9d sequentially or separately by different radiation stimuli. The caged nucleic acid of this invention can also be used either in vivo or in vitro for tracing post-transcriptional and/or post-translational events as well as for blockade of insertion of DNA or RNA intercalating dyes (e.g., pico green; ethidium bromide).
Because expression of the nucleic acid of this invention can be reversibly inhibited, the caged nucleic acid of this invention can be used to regulate expression of an exogenous nucleic acid within a cell. Thus, the present invention further provides a method of selectively expressing a nucleic acid in a cell, comprising: a) covalently linking the nucleic acid to a photolabile caging group which reversibly prevents expression of the nucleic acid; b) introducing the nucleic acid of step (a) into the cell; and c) exposing the cell of step (b) to light, whereby exposure to the light unlinks the nucleic acid and the caging group and the nucleic acid is selectively expressed in the cell. As used herein, xe2x80x9ccovalently linking or linkedxe2x80x9d means forming a covalent bond between the caging group and the target molecule.
The present invention also provides a method of selectively regulating the expression of an endogenous nucleic acid comprising: a) covalently linking an antisense nucleic acid or a nucleic acid encoding an antisense nucleic acid to a photolabile caging group which reversibly prevents expression of the nucleic acid; b) introducing the nucleic acid of step (a) into the cell; and c) exposing the cell of step (b) to light, whereby exposure to the light unlinks the nucleic acid and the caging group and the nucleic acid is present in the cell as an antisense nucleic acid or is expressed in the cell as an antisense nucleic acid which can bind to and inactivate a complementary endogenous nucleic acid within the cell.
Antisense technology is well known in the art and describes a mechanism whereby a nucleic acid comprising a nucleotide sequence which is in an a complementary, xe2x80x9cantisensexe2x80x9d orientation with respect to a coding or xe2x80x9csensexe2x80x9d sequence of an endogenous gene is introduced into a cell, whereby a duplex forms between the antisense sequence and its complementary sense sequence. The formation of this duplex results in inactivation of the endogenous gene. Antisense nucleic acid can be produced for any endogenous gene for which the coding sequence has been or can be determined according to well known methods.
Antisense nucleic acid can inhibit gene expression by forming an RNA/RNA duplex between the antisense RNA and the RNA transcribed from a target gene. The precise mechanism by which this duplex formation decreases the production of the protein encoded by the endogenous gene most likely involves binding of complementary regions of the normal sense mRNA and the antisense RNA strand with duplex formation in a manner that blocks RNA processing and translation. Alternative mechanisms include the formation of a triplex between the antisense RNA and duplex DNA or the formation of a DNA-RNA duplex with subsequent degradation of DNA-RNA hybrids by RNAse H. Furthermore, an antisense effect can result from certain DNA-based oligonucleotides via triple-helix formation between the oligomer and double-stranded DNA which results in the repression of gene transcription.
The antisense nucleic acid may be obtained by any number of techniques known to one skilled in the art. One method of constructing an antisense nucleic acid is to synthesize a recombinant antisense DNA molecule. For example, oligonucleotide synthesis procedures are routine in the art and oligonucleotides coding for a particular protein or regulatory region are readily obtainable through automated DNA synthesis. A nucleic acid for one strand of a double-stranded molecule can be synthesized and hybridized to its complementary strand. One can design these oligonucleotides such that the resulting double-stranded molecule has either internal restriction sites or appropriate 5xe2x80x2 or 3xe2x80x2 overhangs at the termini for cloning into an appropriate vector. Double-stranded molecules coding for relatively large proteins or regulatory regions can be synthesized by first constructing several different double-stranded molecules that code for particular regions of the protein or regulatory region, followed by ligating these DNA molecules together. Once the appropriate DNA molecule is synthesized, this DNA can be cloned downstream of a promoter in an antisense orientation. Techniques such as this are routine in the art and are well documented.
An example of another method of obtaining an antisense nucleic acid is to isolate that nucleic acid from the organism in which it is found and clone it in an antisense orientation. For example, a DNA or cDNA library can be constructed and screened for the presence of the nucleic acid of interest. Methods of constructing and screening such libraries are well known in the art and kits for performing the construction and screening steps are commercially available (for example, Stratagene Cloning Systems, La Jolla, Calif.). Once isolated, the nucleic acid can be directly cloned into an appropriate vector in an antisense orientation, or if necessary, be modified to facilitate the subsequent cloning steps. Such modification steps are routine, an example of which is the addition of oligonucleotide linkers which contain restriction sites to the termini of the nucleic acid. General methods are set forth in Sambrook et al. (1989).
The cell of this invention can be any cell which can incorporate and/or express exogenous nucleic acid and can be exposed to a form of radiation which allows for uncaging of the caging group. For example, the cell of this invention can be, but is not limited to, endothelial cells, epithelial cells and blood cells, as well as any other cell or population of cells (e.g., in an organ or tissue) in which nucleic acid could be selectively expressed according to the methods provided herein. In particular, the method of this invention can be used to selectively express nucleic acid in cells which are easily accessible such as lymphocytes, skin cells and eye cells as well as cells which are internally located (e.g., lung cells accessible by fiber optics) to treat specific disorders associated with these cell types.
In the method of this invention, the nucleic acid can be delivered to the cells in vivo and/or ex vivo by a variety of mechanisms well known in the art (e.g., uptake of naked DNA, viral infection, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis and the like).
If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art. The caged nucleic acid of this invention can be introduced into the cells via any gene transfer mechanism, such as, for example, virus-mediated gene delivery, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes. The transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or homotopically transplanted back into a subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.
For in vivo administration, the cells can be in a subject and the nucleic acid can be administered in a pharmaceutically acceptable carrier. The subject can be any animal in which it is desirable to selectively express a nucleic acid in a cell. In a preferred embodiment, the animal of the present invention is a human. In addition, non-human animals which can be treated by the method of this invention can include, but are not limited to, cats, dogs, birds, horses, cows, goats, sheep, guinea pigs, hamsters, gerbils and rabbits, as well as any other animal in which selective expression of a nucleic acid in a cell can be carried out according to the methods described herein.
In the method described above which includes the introduction of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), the nucleic acids of the present invention can be in the form of naked DNA or the nucleic acids can be in a vector for delivering the nucleic acids to the cells for expression of the nucleic acid inside the cell. The vehicle can be a commercially available preparation, such as an adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the nucleic acid or vehicle to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, Wis.), as well as other liposomes developed according to procedures standard in the art. In addition, the nucleic acid or vehicle of this invention can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.).
As one example, vector delivery can be via a viral system, such as a retroviral vector system which can package a recombinant retroviral genome (Pastan et al., 1988; Miller et al., 1986). The recombinant retrovirus can then be used to infect and thereby deliver nucleic acid to the infected cells. The exact method of introducing the nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are widely available for this procedure including the use of adenoviral vectors (Mitani et al., 1994), adeno-associated viral (AAV) vectors (Goodman et al., 1994), lentiviral vectors (Naidini et al., 1996) and pseudotyped retroviral vectors (Agrawal et al., 1996). Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanisms (see, for example, Schwarzenberger et al., 1996). This invention can be used in conjunction with any of these or other commonly used gene transfer methods.
The nucleic acid and the nucleic acid delivery vehicles of this invention, (e.g., viruses; liposomes, plasmids, vectors) can be in a pharmaceutically acceptable carrier for in vivo administration to a subject. By xe2x80x9cpharmaceutically acceptablexe2x80x9d is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vehicle, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
The nucleic acid or vehicle may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like. The exact amount of the nucleic acid or vehicle required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity or mechanism of any disorder being treated, the particular nucleic acid or vehicle used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every nucleic acid or vehicle. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein (see, e.g., Martin, Remington""s Pharmaceutical Sciences).
As one example, if the nucleic acid of this invention is delivered to the cells of a subject in an adenovirus vector, the dosage for administration of adenovirus to humans can range from about 107 to 109 plaque forming units (pfu) per injection, but can be as high as 1012 pfu per injection (Crystal, 1997; Alvarez, 1997).
Parenteral administration of the nucleic acid or vehicle of the present invention, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Pat. No. 3,610,795, which is incorporated by reference herein.
The present invention additionally provides a polypeptide covalently linked to a photolabile caging group which prevents biological activity of the polypeptide. The polypeptide of this invention can be, but is not limited to, a phosphoprotein which regulates specified patterns of cell division and protein expression, a member of the AP-1 transcription family, a target protein within the MAP kinase cascade and a cyclin, which regulates cell cycle progression. Any protein involved in the cell""s genetic process can be purified can be caged and photoactivated within a cell according to the present invention to regulate cell function in a temporally and spatially dependent manner.
For example, phosphorylated proteins can be photocaged, introduced into cells and targeted to exert an effect in a particular sub-population of cells in a temporally and spatially dependent manner by photolytic uncaging of the caged proteins. One particularly useful phosphoprotein which has been shown to be important in regulating protein expression in endothelial cells is the endogenous inhibitor of the NfkB system called IkB, or the inhibitor of the kB system. This protein normally binds to the NFkB heterodimer (p50, p65) or homodimer (p50, p50) and prevents the nuclear translocation of this transcription factor. The activation of IkB is mediated by its phosphorylation, which causes it to be released from IkB and allows the activation of the complex. Therefore, introduction of a photoactivatable form could be used to regulate this expression.
The caged protein of this invention can be produced according to the protocols described herein. The caged protein can be introduced into cells via a variety of mechanisms well known in the art for delivering proteins to the cytoplasm of a cell. For example, the cage protein can be introduced into the cell via liposome delivery, gene gun delivery, direct injection, endocytosis of a protein which binds the cell surface and the like.
As one example, the caged protein of this invention can be a fusion protein which comprises a caged polypeptide and a ligand which binds to and is internalized by cells which express a receptor for the ligand on the surface.
The ligand of the fusion protein can be any ligand which has the capability of binding to and becoming internalized by cells which express a receptor for the ligand, as determined by methods well known in the art. In this manner, the fusion protein of this invention can be targeted for internalization by specific cell populations which express the receptor which binds the ligand of the fusion protein.
The present invention further provides a nucleic acid encoding the fusion protein of this invention, a vector comprising the nucleic acid and a cell comprising the vector. The present invention also provides nucleic acids complementary to, or capable of, hybridizing with the nucleic acids encoding the fusion proteins of this invention.
Protocols for construction of a vector containing a nucleic acid encoding the fusion protein of this invention are well known in the art (see, e.g., Sambrook et al., 1989). The nucleic acids can be obtained from naturally occurring sources or the nucleic acids can be synthesized. The nucleic acid encoding the fusion protein can be placed into an expression vector, which can be obtained commercially or produced in the laboratory.
A variety of vectors and prokaryotic and eukaryotic expression systems such as bacteria, yeast, filamentous fungi, insect cell lines, bird, fish, transgenic plant and mammalian cells, among others, are known to those of ordinary skill in the art and can be used in the present invention.
Thus, the present invention further contemplates a method of producing the fusion protein of the present invention, comprising introducing a vector encoding the fusion protein into a cell under conditions whereby the nucleic acid encoding the fusion protein is expressed and the fusion protein is produced; and isolating and purifying the fusion protein. Isolation and purification of the fusion protein can be carried out by protocols well known to those of skill in the art.
The nucleic acid sequences can be expressed in cells after the sequences have been operably linked to, i.e., positioned, to ensure the functioning of an expression control sequence. These expression vectors are typically replicable in the cells either as episomes or as an integral part of the cell""s chromosomal DNA. Commonly, expression vectors can contain selection markers, e.g., tetracycline resistance, hygromycin resistance, gentamicin resistance or methotrexate resistance, to permit detection and/or selection of those cells transformed with the desired nucleic acid sequences (see, e.g., U.S. Pat. No. 4,704,362).
The caged protein or fusion protein of the present invention can be administered to cells either ex vivo or in vivo in the same manner as described herein for the caged nucleic acids of this invention. Thus, the caged polypeptide or fusion protein of this invention can be in a pharmaceutically acceptable carrier, as defined herein and can be administered to a subject, which is preferably a human, in accordance with the methods described herein.
Thus, the present invention further provides a method of selectively activating a polypeptide in a cell, comprising: a) covalently linking the polypeptide to a photolabile caging group which reversibly prevents biological activity of the polypeptide; b) introducing the polypeptide of step (a) into the cell; and c) exposing the cell of step (b) to light, whereby exposure to the light unlinks the polypeptide and the caging group and the polypeptide is selectively activated in the cell.
The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.